Protein transduction domains (PTDs) are short peptide sequences that can transport wide varieties of cargo across cell membranes. This study assessed the transduction ability of fusion proteins containing optimised variants of the PTD from HIV-1 transactivator of transcription (Tat). Uptake of Tat-PTDs was determined by fluorescent microscopy using the fluorescent protein Venus as a tag, and also by using fusion proteins containing caspase-7 and RhoA bound to Tat-PTD. Upon entering the cytosol the latter two induce apoptosis and the formation of cytoplasmic extensions, morphological changes easily observed by microscopy. It was found that PTDs with two, three or four sequential Tat-PTD domains could bind to the surface of two of the five cell lines tested. Fluorescent microscopy, however, indicated that the fluorescent constructs remained on the cell surface. As well, PTDs bound to caspase-7 or RhoA did not induce any visible morphological changes in the cells.

Protein transduction domains (PTDs) are short peptide sequences that can transport wide varieties of cargo across cell membranes. This study assessed the transduction ability of fusion proteins containing optimised variants of the PTD from HIV-1 transactivator of transcription (Tat). Uptake of Tat-PTDs was determined by fluorescent microscopy using the fluorescent protein Venus as a tag, and also by using fusion proteins containing caspase-7 and RhoA bound to Tat-PTD. Upon entering the cytosol the latter two induce apoptosis and the formation of cytoplasmic extensions, morphological changes easily observed by microscopy. It was found that PTDs with two, three or four sequential Tat-PTD domains could bind to the surface of two of the five cell lines tested. Fluorescent microscopy, however, indicated that the fluorescent constructs remained on the cell surface. As well, PTDs bound to caspase-7 or RhoA did not induce any visible morphological changes in the cells.

Impaired cell mediated immunity is the clinical hallmark of HIV infection yet the manner in which CD8 T-cells are disabled is not yet fully understood. IL-7 signalling is essential for normal CD8 T-cell development and function. Our lab has previously shown decreased expression of the IL-7 receptor a-chain (CD127) on circulating CD8 T-cells in HIV+ patients is mediated by the HIV Tat protein which results in poor CD8 T-cell function. Soluble Tat protein is secreted by infected CD4 T-cells and taken up by neighbouring uninfected CD8 T-cells through endocytosis. Once in the cytoplasm, Tat translocates to the inner leaflet of the cell membrane where it binds directly to the cytoplasmic tail of CD127 inducing receptor aggregation, internalization, and degradation by the proteasome. By removing CD127 from the cell surface, the HIV Tat protein is able to reduce IL-7 signaling and impair CD8 T-cell proliferation and function. To determine which domain(s) in the cytoplamic tail of CD127 are required for interaction with Tat, a series of plasmids encoding for CD127 deletion mutants were successfully created. These series of mutant CD127 coding sequences were transfected into a eukaryotic expression system, the Jurakt cell line, where CD127 mutants were successfully expressed. Before determine which region on CD127 is required for Tat binding, an optimized Ni-NTA column system was used to successfully isolate histidine-tagged HIV-1 Tat at a high yield and purity from E. coli. This HIV Tat protein was used to treat the lysates of the Jurakt cells transfected with the panel of CD127 mutants. CD127 was then immunoprecipitated, followed by Western analysis of the immune complexes to detect Tat protein. Tat was immunoprecipitated with all CD127 mutants suggests neither tyrosine 449, box 1, the acidic region, serine region nor C-tail are specifically required for Tat binding to CD127.

The HIV-1 transactivator of transcription (Tat) is a protein essential for both viral gene expression and virus replication. Tat is an RNA-binding protein that, in cooperation with host cell factors cyclin T1 and cyclin-dependent kinase 9, regulates transcription at the level of elongation. Tat also interacts with numerous other intracellular and extracellular proteins, and is implicated in a number of pathogenic processes. The Tat protein is encoded by two exons and is 101 residues in length. The first exon encodes a 72-residue molecule that activates transcription with the same proficiency as the full-length protein. The physico-chemical properties of Tat make it a particularly challenging target for structural studies: Tat contains seven cysteine residues, six of which are essential for transactivation, and is highly susceptible to oxidative cross-linking and aggregation. In addition, a basic segment (residues 48-57) gives the protein a high net positive charge of +12 at pH 7, endowing it with a high affinity for anionic polymers and surfaces. In order to study the structure of Tat, both alone and in complex with partner molecules, we have developed a system for the bacterial expression and purification of polyhistidine-tagged and isotopically enriched (in 15N and 15N /13C) recombinant HIV-1 Tat1-72 (BH10 isolate) that yields large amounts of protein. These preparations have facilitated the assignment of 95% of the non-proline backbone resonances using heteronuclear 3-dimensional nuclear magnetic resonance (NMR) spectroscopy. Analysis by mass spectrometry and NMR demonstrate that the cysteine-rich Tat protein is unambiguously reduced and monomeric in aqueous solution at pH 4. NMR chemical shifts and coupling constants suggest that it exists in a disordered conformation. Line broadening and multiple peaks in the cysteine-rich and core regions suggest that transient folding occurs in two of the five sequence domains. NMR relaxation parameters were measured and analysed by spectral density and model-free approaches both confirming the lack of structure throughout the length of the molecule. The absence of a fixed conformation and the observation of fast dynamics are consistent with the ability of the Tat protein to interact with a wide variety of proteins and nucleic acid lending further support to the concept that Tat exists as an intrinsically disordered protein. / October 2007

The HIV-1 transactivator of transcription (Tat) is a protein essential for both viral gene expression and virus replication. Tat is an RNA-binding protein that, in cooperation with host cell factors cyclin T1 and cyclin-dependent kinase 9, regulates transcription at the level of elongation. Tat also interacts with numerous other intracellular and extracellular proteins, and is implicated in a number of pathogenic processes. The Tat protein is encoded by two exons and is 101 residues in length. The first exon encodes a 72-residue molecule that activates transcription with the same proficiency as the full-length protein. The physico-chemical properties of Tat make it a particularly challenging target for structural studies: Tat contains seven cysteine residues, six of which are essential for transactivation, and is highly susceptible to oxidative cross-linking and aggregation. In addition, a basic segment (residues 48-57) gives the protein a high net positive charge of +12 at pH 7, endowing it with a high affinity for anionic polymers and surfaces. In order to study the structure of Tat, both alone and in complex with partner molecules, we have developed a system for the bacterial expression and purification of polyhistidine-tagged and isotopically enriched (in 15N and 15N /13C) recombinant HIV-1 Tat1-72 (BH10 isolate) that yields large amounts of protein. These preparations have facilitated the assignment of 95% of the non-proline backbone resonances using heteronuclear 3-dimensional nuclear magnetic resonance (NMR) spectroscopy. Analysis by mass spectrometry and NMR demonstrate that the cysteine-rich Tat protein is unambiguously reduced and monomeric in aqueous solution at pH 4. NMR chemical shifts and coupling constants suggest that it exists in a disordered conformation. Line broadening and multiple peaks in the cysteine-rich and core regions suggest that transient folding occurs in two of the five sequence domains. NMR relaxation parameters were measured and analysed by spectral density and model-free approaches both confirming the lack of structure throughout the length of the molecule. The absence of a fixed conformation and the observation of fast dynamics are consistent with the ability of the Tat protein to interact with a wide variety of proteins and nucleic acid lending further support to the concept that Tat exists as an intrinsically disordered protein.

The HIV-1 transactivator of transcription (Tat) is a protein essential for both viral gene expression and virus replication. Tat is an RNA-binding protein that, in cooperation with host cell factors cyclin T1 and cyclin-dependent kinase 9, regulates transcription at the level of elongation. Tat also interacts with numerous other intracellular and extracellular proteins, and is implicated in a number of pathogenic processes. The Tat protein is encoded by two exons and is 101 residues in length. The first exon encodes a 72-residue molecule that activates transcription with the same proficiency as the full-length protein. The physico-chemical properties of Tat make it a particularly challenging target for structural studies: Tat contains seven cysteine residues, six of which are essential for transactivation, and is highly susceptible to oxidative cross-linking and aggregation. In addition, a basic segment (residues 48-57) gives the protein a high net positive charge of +12 at pH 7, endowing it with a high affinity for anionic polymers and surfaces. In order to study the structure of Tat, both alone and in complex with partner molecules, we have developed a system for the bacterial expression and purification of polyhistidine-tagged and isotopically enriched (in 15N and 15N /13C) recombinant HIV-1 Tat1-72 (BH10 isolate) that yields large amounts of protein. These preparations have facilitated the assignment of 95% of the non-proline backbone resonances using heteronuclear 3-dimensional nuclear magnetic resonance (NMR) spectroscopy. Analysis by mass spectrometry and NMR demonstrate that the cysteine-rich Tat protein is unambiguously reduced and monomeric in aqueous solution at pH 4. NMR chemical shifts and coupling constants suggest that it exists in a disordered conformation. Line broadening and multiple peaks in the cysteine-rich and core regions suggest that transient folding occurs in two of the five sequence domains. NMR relaxation parameters were measured and analysed by spectral density and model-free approaches both confirming the lack of structure throughout the length of the molecule. The absence of a fixed conformation and the observation of fast dynamics are consistent with the ability of the Tat protein to interact with a wide variety of proteins and nucleic acid lending further support to the concept that Tat exists as an intrinsically disordered protein.

Human immunodeficiency (HIV) infection can result in neurocognitive deficits in about one-half of infected individuals. Despite excellent systemic effectiveness, restricted antiretroviral penetration across the blood-brain barrier (BBB) is a major limitation in fighting HIV infection within the central nervous system (CNS). Drug abuse exacerbates cognitive impairment and pathologic CNS changes in HIV-infected individuals. This work investigates the effects of the HIV-1 protein, Tat, and drugs of abuse on factors affecting drug penetration into the brain. Firstly, an in vitro model of the blood-brain barrier was built to study effects of HIV-1 Tat and methamphetamine (Meth) on integrity and function of the BBB, in turn how HIV-1 Tat and meth will affect antiretroviral penetration into the brain. We found that co-exposure HIV-1 Tat and Meth results in inhibition or impairment of P-glycoprotein activity at the BBB. Also, simultaneous inhibition of P-glycoprotein (P-gp) and Multidrug Resistant Protein -1 (MRP-1), by verapamil and MK-571 causes an increase in accumulation of atazanavir inside the primary human brain endothelial cells. Secondly, we developed and validated the method for simultaneous determination of tenofovir, emtricitabine, and dolutegravir in cell extracts of CNS cells. This method was used to study how HIV-1 Tat and/or morphine affects antiretroviral penetration in CNS cells like human brain microvascular endothelial cells, human astrocytes, human microglia, and human pericytes. We found that in untreated cells, accumulation of antiretroviral drugs was higher in hCMEC/D3 cells compared to other CNS cell types. Also, HIV-1 Tat and/or morphine had no significant effect on antiretroviral penetration amongst these cell types. Overall, the rank order of intracellular accumulation observed in treated and untreated cells was dolutegravir > emtricitabine > tenofovir.

No description available.

These studies were aimed at investigating the potential application of nanoparticles engineered from oil-in-water microemulsion precursors for enhancing immune responses to HIV-1 Tat and Gag p24 proteins. Both of the HIV-1 proteins have been reported to be critical in the virus life cycle and are being evaluated in clinical trials as vaccine candidates. Anionic nanoparticles were prepared using emulsifying wax as the oil phase and Brij 78 and sodium dodecyl sulfate as the surfactants. The resulting nanoparticles were coated with Tat and were demonstrated to produce superior immune responses after administration to BALB/c mice compared to Tat adjuvanted with Alum. Similarly, cationic nanoparticles were prepared using emulsifying wax and Brij 78 and cetyl trimethyl ammonium bromide as the surfactants. The cationic nanoparticles were investigated for delivery of immunostimulatory adjuvants, namely three Toll-like receptor ligands, for obtaining synergistic enhancements in immune responses to a model antigen, Ovalbumin (OVA). In vitro and in vivo studies were carried out to elucidate possible mechanisms by which nanoparticles may result in enhancements in immune responses. In vitro studies were carried out to evaluate the uptake of nanoparticles into dendritic cells and to assess the release of pro-inflammatory cytokines from dendritic cells in the presence of nanoparicles. In vivo studies were carried out using a MHC class I restricted transgenic mouse model to investigate the potential for nanoparticles coated with OVA to enhance presentation of the protein to CD8+ T cells compared to OVA alone. Finally, the preparation of nanoparticles with a low amount of surface chelated nickel for high affinity binding to histidine-tagged (his-tag) proteins was investigated. It was hypothesized that this strengthened interaction of his-tag protein to the nickel chelated nanoparticles (Ni-NPs) would result in a greater uptake of antigen in vivo; therefore, enhanced immune responses compared to protein bound to anionic nanoparticles. In vivo evaluation of his-tag HIV-1 Gag p24 bound to Ni-NPs resulted in enhanced immune responses compared to protein either adjuvanted with Alum or coated on the surface of nanoparticles.

More than 1.2 million people are estimated to be currently living with the human immunodeficiency virus (HIV) in the United States of America. The gastrointestinal (GI) tract is both a major target and an important component of HIV pathogenesis. The GI processes that are dysregulated during HIV infection are controlled by the enteric nervous system (ENS). Indeed, both clinical and experimental studies have implicated the ENS in HIV and simian immunodeficiency virus (SIV) pathogenesis. In addition to direct viral effects, the HIV virus also indirectly affects the GI tract via cellular and/or viral toxins released by infected cells. Trans-activator of transcription (Tat) is a viral toxin that plays an important role in replication of the HIV virus. While, the HIV virus does not directly infect neurons, Tat has been shown to modulate neuronal function. HIV infection in the gut is accompanied by: translocation of bacteria and bacterial products from the gut lumen to peripheral blood, immune activation and inflammation. Lipopolysaccharide (LPS) is a major bacterial product that is used to determine the rate of bacterial translocation and to drive inflammation. Despite reports of enteric ganglionitis in SIV infected monkeys and autonomic denervation in the jejunum of HIV patients, little is known of the mechanism underlying enteric neuropathogenesis in HIV and the role of the ENS in HIV pathogenesis. In the present study, we assessed the effects of Tat on enteric neuronal excitability and how Tat and LPS interact in the ENS to bring about inflammation and GI motility problems observed in HIV patients. We show that Tat significantly increased enteric neuronal excitability by modulating sodium channels expressed on enteric neurons. Tat sensitized ENS cells to LPS-mediated increase in pro-inflammatory cytokines via a TLR4-mediated pathway involving MyD88. Mice expressing the tat transgene (Tat+) had faster GI transit rates and significantly higher frequencies of diameter changes in the proximal ileum than controls (Tat-). Tat+ mice were also more sensitive to LPS-mediated decreases in colonic transit rate. This study highlights the role of viral and bacterial proteins in HIV pathogenesis in the gastrointestinal tract and also demonstrates a critical role of the ENS in HIV pathogenesis.